1. Field of the Invention
The present invention relates to compositions and methods for high level expression of polypeptides. The invention provides nucleic acid molecules, expression systems, host cells, methods and kits that are useful for the production of polypeptides and/or untranslated RNA molecules. Due to the improved expression levels of polypeptides or untranslated RNA molecules the invention is particularly useful for the rapid production of large quantities of recombinant proteins such as monoclonal antibodies which can be used as drugs.
2. Related Art
Process development for biopharmaceuticals, and hereby in particular the expression of polypetides used in these biopharmaceuticals, is governed by the economy of the manufacturing process. The classical approach for the production of recombinant polypetides is the use of stable expression systems. These systems are based on chromosomal integration of an expression plasmid into the genome of the host cell. The sites of gene integration, however, are random, and the number and ratio of genes integrating at any particular site are unpredictable. As a result, it is normally necessary to screen many clonal cell populations to obtain a cell line in which the desired genes are expressed at an appropriate level. This procedure of transfection, selection and analysis of numerous clonally derived cell lines expressing the multiple genes is costly and time-consuming.
An alternative to stable expression systems for gene expression are transient expression systems. The expression of the latter systems is based on non-integrated plasmids, and hence the expression is typically rapidly lost as the cell undergoes division. Thus, transient expression systems typically do not lead to sufficient expression over time implying that repeated processes would be necessary which might be not desirable.
Another way of performing expression is by transfection of an episomal vector which replicates extra-chromosomally in a host cell such as in a mammalian host cell. Episomal replicating vectors provide certain advantages over classical expression systems since they do not integrate into the host genome, but replicate episomally in a host cell. Most of the described episomally replicating vectors are based on viral components from viruses like Papovaviridae (e.g. SV40, BPV) or Herpesviridae (e.g. EBV). Episomal replicating vectors derived from these viruses generally contain a replication origin and at least one viral trans-acting factor, e.g., an initiator protein. Examples of such initiator proteins are large T-antigen for SV40, E1/E2 for BPV, and EBNA-1 for EBV. The process of episomal replication typically involves both host cell replication machinery and virus trans-acting factors.
To increase expression, retrovirus vectors stably integrated in transduced resting cells have been described using a chromosomal scaffold/matrix attached regions (S/MAR) element derived from human INF-beta gene (U.S. Pat. No. 6,194,212 B1). It has further been shown for stably integrated retrovirus vectors that human INF-beta SAR inhibits de novo methylation, alleviates methylation-mediated transcriptional repression and allows for higher level gene expression (Dang et al., (2000) J Virol 74, 2671-2678). Episomal vectors having a S/MAR element were described for delivery in gene therapy. However, to avoid any type of cell transformation induced by viral trans-acting factors, the viral trans-acting factor was replaced by S/MAR (U.S. Pat. No. 6,410,314 B1; Piechaczek et al., NAR, 1999, Vol 27, No. 2; Bode et al., 2001 Gene Ther Mol Biol, Vol. 6, 33-46).
However, the enhancing effect of S/MAR on promoter functions and gene expression has been reported to be restricted to the integrated state of transfected templates. Thus, in transient expression systems for the expression of proteins, and therefore in expression systems based on plasmids which are not integrated in the genome of the host cell, the effect of S/MAR on the expression levels of proteins has been reported to have an antagonizing effect (Klehr et al., (1991) Biochemistry, 30(5):1264-70; Poljak et al., (1994) Nucleic Acid Res., 22(21):4386-94; Wu et al., (2001) Sheng, 33(1):59-64).
A need remains in the art for compositions and methods other than stable expression systems that allow rapid production of large quantities of polypeptides and untranslated RNA molecules, and in particular for compositions and methods that allow increased expression levels of a desired protein compared to compositions and methods known in the art.